A measured difference in RNA expression level between two samples is the result of both true biological as well as experimentally induced (technical) variation. Different variables, inherent to the RT-qPCR workflow need to be controlled for in order to minimize the technical variation. Influencing parameters include the amount and quality of starting material, enzymatic efficiencies, and overall transcriptional activity.

It is highly recommended to minimize the technical variation by using standard operating procedures throughout the entire qPCR workflow. The remaining technical variation should then be further reduced or removed by using a proper normalization approach, enabling a better appreciation of the true biological variation.

Back in 2002 ( ), we pioneered a new normalization strategy for more accurate normalization of mRNA expression data from RT-qPCR studies. Since then, the use of multiple stably expressed reference genes has become the gold standard method (see also MIQE guidelines in ). Today, more than 11000 papers have cited our seminal paper (according to Google Scholar)!